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© Marion Coolen
Radial glia neural stem cells of the adult zebrafish telencephalon electroporated with a membrane tagged GFP
Publication : Light, science & applications

Dual-color deep-tissue three-photon microscopy with a multiband infrared laser

Scientific Fields
Diseases
Organisms
Applications
Technique

Published in Light, science & applications - 06 Jun 2018

Guesmi K, Abdeladim L, Tozer S, Mahou P, Kumamoto T, Jurkus K, Rigaud P, Loulier K, Dray N, Georges P, Hanna M, Livet J, Supatto W, Beaurepaire E, Druon F

Link to Pubmed [PMID] – 30839589

Light Sci Appl 2018;7:12

Multiphoton microscopy combined with genetically encoded fluorescent indicators is a central tool in biology. Three-photon (3P) microscopy with excitation in the short-wavelength infrared (SWIR) water transparency bands at 1.3 and 1.7 µm opens up new opportunities for deep-tissue imaging. However, novel strategies are needed to enable in-depth multicolor fluorescence imaging and fully develop such an imaging approach. Here, we report on a novel multiband SWIR source that simultaneously emits ultrashort pulses at 1.3 and 1.7 µm that has characteristics optimized for 3P microscopy: sub-70 fs duration, 1.25 MHz repetition rate, and µJ-range pulse energy. In turn, we achieve simultaneous 3P excitation of green fluorescent protein (GFP) and red fluorescent proteins (mRFP, mCherry, tdTomato) along with third-harmonic generation. We demonstrate in-depth dual-color 3P imaging in a fixed mouse brain, chick embryo spinal cord, and live adult zebrafish brain, with an improved signal-to-background ratio compared to multicolor two-photon imaging. This development opens the way towards multiparametric imaging deep within scattering tissues.

https://www.ncbi.nlm.nih.gov/pubmed/30839589